The broad long term objective of this proposal is to determine the molecular basis of superoxide production in human neutrophils. The proposed investigation will focus on the structure of human neutrophil cytochrome b and how it changes upon activation of the superoxide generating system. The fundamental assumption made in this proposal is that this cytochrome is the central electron transferase of superoxide production and that modifications of its structure will regulate the flow of electrons across the plasma membrane. Understanding the molecular mechanisms of regulation of this electron flow will provide crucial information necessary for a molecular understanding of microbicidal killing and misdirected tissue injury functions of human neutrophils. Hence, understanding the structure of this cytochrome may lead to the development of rationally designed drugs that could ameliorate neutrophil mediated tissue damage and enhance neutrophil mediated microbicidal killing. More specifically, this proposal outlines strategies for making monoclonal antibodies recognizing native cytochrome b and defining the binding epitopes of these antibodies using a random sequence phage library. It describes a plan to covalently modify cytochrome b in order to mark different sites on its surface with fluorescent probes, identify possible phosphorylation sites, and identify the locations of the heme prosthetic groups. To precisely identify these modifications, it proposes to use HPLC combined with electrospray mass spectrometry and peptide sequencing of proteolytic digests of the modified proteins. Using this information and covalent modification of the cytochrome with fluorescent probes, it then should be possible to map the cytochrome surface relative to intrinsic hemes using fluorescence energy transfer as a spectroscopic ruler. Lastly the proposal describes a strategy to use multidimensional NMR techniques employing TOCSY, ROSEY, and Transferred NOESY to determine the bound structures of interfacial sites of the cytochrome that interact with its functional partners m the superoxide-generating NADPH oxidase. Successful completion of this work will permit the development of a structural model of the cytochrome incorporating sequence information, transmembrane topology, locations of specific sites, and high resolution maps of interfacial regions.